Stem Cell

Saxitoxin potentiates human neuronal cell death induced by Zika virus while sparing neural progenitors and astrocytes – Scientific Reports

Time Stamp: September 30, 2024 8:00 PM
Source Node: 680283

Human induced pluripotent stem cells

All cell types used in the present work were derived from human induced pluripotent stem cells (hiPSCs) obtained after reprogramming somatic cells from three different healthy individuals. Two of them were derived from fibroblasts isolated from skin biopsies. The third one (GM23279A) is commercially available at the NIGMS Human Genetic Cell Repository, obtained and certified by the Coriell Institute for Medical Research cell bank. All in-house hiPSCs were reprogrammed using a previously published protocol⁴⁵ and were used in subsequent works by our group^{17,46,47,48,49}. Written informed consent was obtained from the subjects who provided their samples for iPSC reprogramming and ethical approvals were attained by the ethics committee of Copa D’Or Hospital (CAAE number 60944916.5.0000.5249, approval number 1.791.182). All human cell experiments were performed in accordance with Copa D’Or Hospital regulations. All cells grown in monolayers, Geltrex LDEV-Free (Thermo, A1413302) and StemFlex medium (Thermo, A3349401), used in this work were maintained at 37 °C, in an atmosphere containing 5% CO₂ and cultivated in the absence of antibiotics.

Neural stem cells (NSCs)

Neural Stem Cells (NSCs) were generated from hiPSCs cells following the published Life Technologies protocol for “Induction of Neural Stem Cells from Human Pluripotent Stem Cells” (Publication number: MAN0008031). Briefly, the hiPSCs were maintained for 7 days in a medium containing Neurobasal (Thermo Fisher Scientific, 12348017) and 1% neural induction supplement (NIS – Life Technologies, a16477-01) which was changed every day on an adherent plate coated with Geltrex LDEV-Free (Thermo, A1413302). After induction, NSCs were grown in adherent 60 mm plates coated with Geltrex LDEV-Free and in NEM medium (Neural Expansion Medium), composed of 49% Neurobasal (Thermo Fisher Scientific, 12348017), 49% Advanced-DMEM (Thermo Fisher, 12634-010) and 2% neural induction supplement (Life Technologies, A16477-01). The medium was changed every 2 days and when 90–100% confluence was reached the cells were enzymatically dissociated with Accutase (Millipore, SCR005). For experiments, cells were plated onto 96-well plates at a density of 10,000 cells per well or in 24-well plates with glass coverslips at a density of 40,000 cells per well, both plates previously coated with Geltrex.

Glial differentiation

Astroglial induction was based on the protocol previously described by Yan et al.^50,51. The protocol started with NSC plated at 1.25 × 10⁶ in a T-25 flask, previously coated with 1x Geltrex (Thermo, A1413302). On the day after plating, NEM medium was replaced by AIM medium (Astrocyte Induction Medium) composed of DMEM/F12 (Life Technologies, 11330-032), 1% Fetal Bovine Serum (FBS – Thermo, 12657029) and 1x N2 (Invitrogen, 17502001). The AIM medium was changed every 2 days for 21 days. During these first 21 days, cell cultures were passed with Accutase when 90% confluence was reached and split 1:4. On the 14th day the coating of the flasks was made with a reduced concentration (0.5x) of Geltrex. After day 21, an astrocyte expansion medium consisting of DMEM/F12 with 10% FBS was added, and the cells passed to uncoated flasks. Cells were maintained for at least 6 weeks in this medium for further maturation. Glial cells between 6 and 20 weeks of maturation were used for the assays. For experiments, the cells were plated in 96 well plates with 2.5 × 10³ cells per well or in 24-well plates, containing glass coverslips, with a density of 2.5 × 10⁴ cells per well.

Neuronal differentiation

Human cortical neurons

The neuronal differentiation protocol was adapted from two previously described protocols⁵⁰. To obtain a mixed neuronal culture, 3.0 × 10⁶ NSCs were first seeded in a 100 mm plate, previously coated with 10 µg/mL laminin (Invitrogen, 23017-015) in NEM medium. The next day, the medium was changed to neuronal differentiation medium, which consists of Neurobasal (Life Technologies, 21103049), 1x B27 Supplement (Life Technologies, 17504044), 1x Glutamax (Life Technologies, 35050061), 1x non-essential amino acids (Life Technologies, 11140050) and 200 µM ascorbic acid (AA) (Sigma Aldrich, A4034). This medium was maintained until the end of the experiment, being changed every other day. On the 7th and 14th days, the cells were passed with Accutase to a plate coated with 10 µg/mL laminin at least 3 h before, at 37 °C, and kept until the neurons completed 50 days. For experiments, cells were plated in 96-well plates with 3 × 10⁴ cells per well or in 24-well plates, containing glass coverslips, with a density of 2 × 10⁵ cells per well. For the electrophysiological recordings, neuronal stem cells (NSC) were plated in glass microelectrode array (MEA) chips from Multi Channel Systems (product reference 60MEA200/30iR-Ti) coated with 500 µg/mL poli-ornithine and 20 µg/mL laminin and differentiated into neuronal cultures in neuronal differentiation medium for 45 days, with culture medium changes every two-three days. Cortical neurons grown on MEA were exposed to saxitoxin for 10 days at 12 ng/mL beginning at day 45, with repeated additions every change of medium (every 3–4 days).

Human sensory neurons

The sensory neuron differentiation protocol was performed following some modifications from the published protocol by Guimarães et al.²². Briefly, NSCs (5 × 10⁶) were seeded in NEM medium onto 100 mm plates coated with 100 µg/mL polyornithine (PLO) (Sigma Aldrich, P3655) and 20 µg/mL laminin. Next day, the medium was changed to 3 N for neuronal differentiation (DMEM-F12, Neurobasal Medium, 1x Glutamax, 0.5x N2 Supplement, 0.5x B27 Supplement, 0.5x non-essential amino acids, and 1:1000 β- mercaptoethanol (Sigma Aldrich, M3148) supplemented with 10 ng/mL BDNF (R&D systems, 248-BD-025), 200 µM Ascorbic Acid (AA) (Sigma Aldrich, A4034), 10 ng/mL GDNF (R&D Systems, 212-GD-010), 10 ng/mL NGF (R&D Systems, 256- GF-100),10 ng/mL NT-3 (R&D Systems, 267-N3-025) and 0.5 mM cAMP (Sigma-Aldrich, D0260-100MG). Medium changes were performed twice a week. On day 45, cells were detached with Accutase and incubated with the same neuronal medium supplemented with 10 µM iRock (Millipore) and plated for experiments. Cells were seeded in 96-well plates with 3 × 10⁴ cells per well or in 24-well plates, containing glass coverslips, with a density of 2 × 10⁵ cells per well.

Brain organoids

The production of organoids followed a protocol previously described by Goto-Silva et al.⁵², in which hiPSCs were cultivated in mTeSR1 medium (StemCell Technologies, 05850) on Matrigel (BD Biosciences, CLS354277). When colonies reached 70–80% confluency, hiPSCs were dissociated with Accutase and seeded at 9,000 cells/well into 96-well round bottom low attachment plates (Corning, 7007) in hiPSCs medium. The next day, medium was replaced with hESC medium (85% DMEM/F12, 10% knockout serum replacement (KOSR), 3% ESC-quality FBS, 1% Pen-Strep, 1% GlutaMAX, 1% MEM-NEAA, 0.7 µL/mL 2-mercaptoethanol), and the embryoid bodies (EBs) were cultured for 6 days in the hESC medium previously described in Lancaster et al.. (2014)⁵³. On day 6 the EBs were transferred to 24-well flat bottom ultra-low attachment culture plates (Corning, 3473) containing Neural Induction Medium, DMEM/F12, 1% Pen-Strep (Thermo Fisher Scientific, 15140122), 1% N2, 1% GlutaMAX, 1% MEM-NEAA and 1 µg/mL heparin (Thermo Fisher Scientific, H3149-100KU) for 4 days. The organoids were coated with Matrigel for 1 h at 37 °C and 5% CO₂ and returned to 24-well flat bottom ultra-low attachment plates. Organoids were maintained for 4 days in static culture in neural differentiation medium, DMEM/F12, and Neurobasal medium (1:1), 1% Pen-Strep, 0.5% N2, 0.5% B-27 without vitamin A (Thermo Fisher Scientific, 17504044), 1% GlutaMAX, 0.5% MEM-NEAA, 0.035% 2-mercaptoethanol, and 1:4000 insulin (Sigma Aldrich, I9278) and after that time were cultured in suspension in 6 wells plates, under constant stirring at 90 rpm on an orbital shaker. For this final step, 4–6 organoids were allocated per well containing 3 mL of neural differentiation medium with added vitamin A (only changing the B27 supplement to Thermo Fisher Scientific, 17504001). The medium was changed twice a week until completing 45 days of agitation when they were used for experiments.

Replication and viral infection

The Asian viral strain (AS) ZIKV (Recife/Brazil, ZIKV PE/243, number: KX197192.1) was provided by Dr. Marli Tenório Cordeiro from Fundação Oswaldo Cruz/Centro de Pesquisas Aggeu Magalhães, Brazil. The virus was propagated in the C6/36 Aedes albopictus cell line at a multiplicity of infection (MOI) of 0.01 and cultured for 6 days in Leibovitz’s L-15 medium (Thermo Fisher Scientific, 11415064) supplemented with 0.3% tryptose phosphate broth (Sigma-Aldrich, T4532), 2 mM L-glutamine (Thermo Fisher Scientific, 25030081) and 1x MEM non-essential amino acids and 2% FBS. ZIKV titers were determined by conventional plaque assay.

NSCs, astrocytes, and neurons were treated with saxitoxin for 1 week at 6 ng/mL (neurons only) or 12 ng/mL (all cell types). After that, cells were washed once with 1x PBS and incubated with the viral inoculum for 2 h at an MOI = 0.5. In the case of astrocytes, FBS was withheld from the infection solution, to maximize viral action. Infection controls were incubated with the supernatant of uninfected C6/36 Aedes albopictus cell line (MOCK). After this time, the viral inoculum was removed, and the cells were incubated for another 72 h in their respective culture media containing saxitoxin. Brain organoids were infected for 2 h at MOI 0.5 and then maintained for up to 13 days. Saxitoxin was added at a concentration of 12 ng/mL immediately after the 2 h of infection and added at each medium change throughout the 13 days.

MTT cell viability assay

After 72 h of infection, 10 µL of 0.5 mg/mL MTT (3-(4,5-dimetiltiazole-2yl)-2.5- diphenyl tetrazolium), diluted in DMEM/F12, was added per well and the cells were incubated for 3 h at 37 °C. Following this period, the MTT solution was removed and 100 µL of DMSO was added per well. Then, the absorbances at wavelengths of 560 nm and 690 nm were determined in the Tecan Infinite 200 PRO plate reader. The absorbance was corrected by calculating the difference between values obtained at 560 nm and the reference wavelength (690 nm). The relative viability was calculated considering the untreated uninfected group as 100% viability.

Immunofluorescence analysis

Cells were fixed in 4% paraformaldehyde (PFA) for 20 min at 37 °C after 72 h infection ZIKV. The cells were washed 3 times with 1x PBS and incubated for 2 h with 5% normal goat serum (NGS – Sigma- Aldrich, G9023) in 1x PBS with 0.3% Triton (Sigma-Aldrich, T8787). Then, the primary antibodies [Non-structural protein 1 from ZIKV (NS1, 1:500, BioFront Technologies, BF1225-06;), Class III β-tubulin (TuJ3, 1:200, Sigma-Aldrich, t3952), MAP2 (1:200, Thermo Fischer Scientific PA517646), and TRPV1 (1:250, Abcam, ab3487) were diluted in the same solution and incubated overnight at 4 °C. The next day, coverslips were washed 3 times with PBS 1x, and incubated with secondary antibodies AlexaFluor 488 (1:400, Thermo Fischer Scientific A-11008 and A-11001) and AlexaFluor 647 (1:400, Thermo Fischer Scientific A31573 and A32728) diluted in PBS for 2 h at room temperature, after which they were counterstained with 300 nM 4’,6’-diamino-2-phenylindole (DAPI; 10236276001, Roche) for 5 min, the coverslips were rewashed with 1x PBS and milli-Q water and finally mounted with AquaMount (Polysciences, 18606-20). To assess cell death, the Click-iT™ TUNEL Alexa Fluor™ 594 Imaging Assay was used (Thermo Fisher Scientific, C10246) following the instructions provided by the manufacturer. All images were acquired on a Leica TCS SP8 confocal microscope. Infection was quantified by counting the number of NS1 positive cells per field compared to the total number of cells (DAPI positive), by analyzing the perinuclear NS1 staining. Cell death was quantified by counting the number of tunnel-positive cells per field compared to the total number of cells (DAPI positive). At least 10 fields per coverslip (average of 2 coverslips per experimental group) were quantified and 3 independent experiments were performed. Quantification was obtained using the Cell Counter plugin of the ImageJ software.

Isotropic fractionation

ZIKV-infected organoids treated with saxitoxin were fixed and the nuclei of these organoids were obtained by isotropic fractionation following the protocol in Herculano and Lent¹⁸. These nuclei were seeded in 384 plates coated with 0.1 mg/mL poly-L- lysine. Cell death was detected by the Click-iT™ TUNEL Alexa Fluor™ 594 Imaging Assay and staining was performed according to the manufacturer’s instructions. Antibodies NeuN (1:100, Millipore, MAB377) and TBR1 (1:200, Thermo Fisher Scientific, PA530971) were used to stain neuronal nuclei, of mature and immature neurons, respectively. The nuclei were labeled with 0.5 µg/mL of 4’-6’-diamino-2-phenylindole (DAPI) for 10 min. Nuclei were washed with 1x PBS, mounted with glycerol, and analyzed on an Operetta high-content imaging system with a 40x objective and high numerical apertures (NA) (PerkinElmer, USA). Data were analyzed using Harmony 5.1 high-content image analysis software (PerkinElmer, USA), to quantify the number of positive cells per marker, relative to DAPI. Ten independent photomicrographs were evaluated from each of three distinct wells per organoid, using 3 organoids for each experimental condition, obtained in 3 distinct organoid batches.

Electrophysiology

Before the electrophysiological recordings, the culture medium of MEA plates with 45-days-old cultured hiPSC-derived neurons, as specified above, was replaced by a culture medium composed of 50% neuronal differentiation medium and 50% BrainPhys™ neuronal culture medium (StemCell, 05790) with BDNF (10 ng/mL), GDNF (10 ng/mL), ascorbic acid (200 nM) and cAMP (1 mM). After 24 h, MEA plates were placed in the MEA2100 System from Multi Channel Systems (MCS, Reutlingen, Germany) and data recording took place at 37 °C after 3 min of stabilization. All recordings were acquired with the Multi Channel Experimenter software (version 2.20.9.23272) at 20 kHz for 20 min. After each recording, the raw digitized data were analyzed in the Matlab based MEA-Toolbox software (version 1.151)⁵⁴. Signals were digitally filtered by a high-pass Butterworth order 2 filter with a 20 Hz cutoff frequency. The standard deviation (SD) of the filtered signals was calculated separately for each channel. Fast deflections with amplitudes greater than 5 times the SD were considered spikes. Spikes detected within 3 ms of a previously detected spike were eliminated from further analysis. A channel was considered active if the mean firing rate (MFR) was greater than 0.1 Hz. Only MEA chips with at least 14 active channels were used for STX treatments and posterior analysis.

Toxins

Saxitoxin dihydrochloride and Tetrodotoxin were purchased from the National Research Council Canada, kept aliquoted at -20^oC and freshly thawed for each experiment.

Statistical analysis

Data were expressed as the average ± standard error of the mean (SEM). Replica values of the same conditions were submitted to the Grubbs test for outliers, in which values considered significantly discrepant (α = 0.05) were disregarded. For the analyses of significance, we used Two-Way ANOVA (Mixed-effects model) both followed by Tukey’s multiple comparisons tests. The differences were considered statistically significant when p < 0.05.